The regulatory framework of leaf senescence is gradually becoming clearer; however, the fine regulation of this process remains largely unknown.
Here, genetic analysis revealed that U2 small nuclear ribonucleoprotein B (U2B″), a component of the spliceosome, is a negative regulator of leaf senescence. Mutation of U2B″ led to precocious leaf senescence, whereas overexpression of U2B″ extended leaf longevity. Transcriptome analysis revealed that the jasmonic acid (JA) signaling pathway was activated in the u2b″ mutant. U2B″ enhances the generation of splicing variant JASMONATE ZIM-DOMAIN 9β (JAZ9β) with an intron retention in the Jas motif, which compromises its interaction with CORONATINE INSENSITIVE1 and thus enhances the stability of JAZ9β protein. Moreover, JAZ9β could interact with MYC2 and obstruct its activity, thereby attenuating JA signaling. Correspondingly, overexpression of JAZ9β rescued the early senescence phenotype of the u2b″ mutant.
Furthermore, JA treatment promoted expression of U2B″ that was found to be a direct target of MYC2. Overexpression of MYC2 in the u2b″ mutant resulted in a more pronounced premature senescence than that in wild-type plants.
Collectively, our findings reveal that the spliceosomal protein U2B″ fine-tunes leaf senescence by enhancing the expression of JAZ9β and thereby attenuating JA signaling.
Alternative splicing (AS) is a mechanism by which cells generate abundant protein diversity from a limited number of genes (Baralle & Giudice, 2017). AS plays a crucial role in regulating various life activities such as growth, development, and aging in plants (Zhu et al., 2017; Godoy Herz & Kornblihtt, 2019; Jabre et al., 2019; Chen et al., 2020; Reddy et al., 2020; Zhang et al., 2020), where it greatly influences plant growth, development, and response to biotic and abiotic stresses (Motion et al., 2015; Laloum et al., 2018; Chaudhary et al., 2019; Chen et al., 2021; Ganie & Reddy, 2021; Saini et al., 2021; Zhu et al., 2023; Supporting Information Fig. S1). The traditional method for the identification of AS is semi-quantitative RT-PCR, which is easy to perform (Palusa et al., 2007; Li et al., 2020; Riegler et al., 2021; Han et al., 2022). Quantitative PCR (qPCR) is also widely used in AS research, as it enables real-time monitoring of fluorescence signals and accurate quantification of isoform copy numbers through the use of specific primers (Hefti et al., 2018; Liu et al., 2018; Huang et al., 2021). With the emergence of digital PCR (dPCR), the identification methods of AS have become more diversified, which disperses each single target fragment into separate droplets as much as possible through the calculation of positive droplets (Fig. S2; Gao et al., 2021).
Based on the urgent need for the accurate quantification of various isoforms, an AS detection method called QuantAS was established (Fig. 1), which allows us to accurately quantify all isoforms of genes based on absolute quantification technology and specific primer design. The method utilizes isoform-specific primers to overcome the isoform identification difficulty caused by different AS events and is designed by using the functional coding region as the isoform structure classification unit to ensure isoform independence (Fig. 2a). RT-qPCR enables real-time monitoring of changes in the fluorescence signal, quantification of differences between expression levels, and simultaneous detection of multiple in a single reaction. According to the copy number of different isoforms, isoform expression patterns can be identified by combining with absolute quantitative techniques. This method greatly increases the accuracy of identification and reduces the cost of repeated experiments. Furthermore, the absolute quantification of AS isoforms employing the combination of qPCR and dPCR could provide their respective advantages, thus rapidly obtaining all isoform info
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